Life phenomena are a system that is realized by complex interaction among many gene products, and it is an essential as a starting point to accurately grasp the expression dynamics of each gene product for understanding the true nature of the life phenomena. Microarrays are actively used as means for the systematic analysis of gene expression. A microarray analysis is a method in which thousands to tens of thousands of oligonucleotide spots are formed on a support such as a slide glass, a target prepared from RNA derived from cells or tissues to be analyzed are hybridized, and the amount of transcription of each gene is comprehensively measured using the intensity of hybrid formation obtained as an index. This method is useful for efficiently and quantitatively measuring the dynamic behavior of all genes involved in living organisms, whereby the method provides gene expression information in various life phenomena and may be a first step for expanding the method to effective applications in a medical, food industry or substance production field.
A series of oligonucleotide microarray GeneChip (trade name: manufactured by Affymetrix, Inc.) is able to comprehensively and quantitatively measure a gene expression level and becomes almost mainstream. In this method, cDNA is prepared first by reverse transcription of total mRNAs derived from cells or tissues, labeled cRNA (antisense strand) is synthesized by in-vitro transcription reactions using T7 RNA polymerase, and the synthesized cRNA is hybridized on the array. However, 1,000 to 100,000 cells are necessary for extractin the total mRNA to prepare a target.
Since a trace amount of cells comprising an extremely small number of cells are responsible for crucial functions in the generation process of multicellular organisms including human and in nerve cells, blood cells, somatic stem cells and cancer cells, it seems essential to ultimately analyze one cell, or at a single cell level. As an important point of an analysis at the single cell level, it is necessary to amplify the target nucleotide sequence to a level capable of applying to the microarray analysis for a quantitative microarray analysis. It is also necessary to amplify the nucleotide sequence while a relative relation of a gene expression level in the single cell is maintained as much as possible.
A method for synthesizing cDNAs from a quite minute amount of mRNA at the single cell level and for amplifying the cDNAs while the relative relation of gene expression level is maintained had been first proposed by G. Brady et al. in 1990, and has been improved thereafter (Non-patent Documents 1 and 2). Microarray experiments carried out by labeling the cDNAs amplified by this method with fluorescent reagents have been reported in 2003 (Non-patent Document 3). However, these technologies have some defects to be described below, and quantitative a microarray analysis at the single cell level has not been practically used yet.
(1) Distortion of Relative Relation of Gene Expression Level by Usual PCR Method
In a PCR method, DNA is exponentially amplified by doubling a template and further doubling the template. Accordingly, a quite small difference of amplification efficiency among different gene products finally causes a multifold difference, to largely distort the relative relation of the gene expression level. This is serious for the gene products whose expression level is relatively small, and is a dominant cause for relatively large decrease of detection sensitivity of such a gene product. Such distortion of the relative relation of the gene expression level in the amplification process by the usual PCR method is referred to a “systematic error”.
(2) Variation of Gene Expression Level in Amplification Process
Random errors in the amplification process are also exponentially amplified due to exponential amplification characteristics of the PCR method. A small error caused in each step of the amplification process finally generates a severalfold difference. Thereafter, this is a crucial factor for reducing reliability when the expression level of each gene product is estimated after amplification. This is a so-called random error of the expression level of each gene in the amplification process.
In recent years, there is reported, in order to overcome the defects of the PCR method, a method for addition of a 3′-end T7 promoter when synthesizing a primary chain of cDNA from a minute amount of mRNA at the single cell level (in case of synthesis of a primary cDNA chain) to perform amplification (linear amplification) by an in vitro transcription reaction (Non-patent Document 4). A kit taking advantage of this principle has been commercialized (sales agent: EPICENTRE Biotechnologies). However, a work for extracting RNAs from a single cell is necessary for the oligonucleotide microarray experiments for amplifying a minute amount of mRNA at the single cell level by using this kit while the relative relation of the gene expression level is maintained. However, there is not practical to extract and purify a minute amount of RNA at this level, and it cannot be denied that a low copy mRNA may be lost in this work. In addition, since the amplification efficiency of the nucleotide sequence by linear amplification is extremely inferior to the amplification efficiency by the PCR method, the amount of the amplification products to be obtained is small. Further, since only one microarray experiment can be performed in one amplification experiment and the amplification product is inevitably labeled RNA in the linear amplification, and preservation of the sample is difficult as compared with DNA samples. It is inconvenient that a reverse transcription reaction is necessary again before gene-specific PCR for confirming whether amplification has been succeeded or not because the amplification product is labeled RNA. Furthermore, since high level of skill is necessary for complicated experimental works in which synthesis and purification of RNA are repeated, this method seems to be not suitable for an analysis at an actual single cell level.
Non-patent Document 1: Brady, G., M. Barbara et al. (1990), “Representative in vitro cDNA amplification from individual henopoietic cells and colonies”, Methods Molec. Cell. Biol. 2 (17-25)
Non-patent Document 2: Iscove, N. N., M. Barbara, et al., (2002), “Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA”, Nat Biotechnol 20(9): 940-3
Non-patent Document 3: Tietjen, I., J. M. Rihel, et al. (2003), “Single-cell transcriptional analysis of neuronal progenitors”, Neuron 38(2): 161-75
Non-patent Document 4: Kamme et al., “Single-Cell Microarray Analysis in Hippocampus CAI: Demonstration and Validation of Cellular Heterogeneity”, The Journal of Neuroscience, May 1, 2003, 23(9):3607